Oxidized LDL as a biomarker for neurological complications of pregnancy

ABSTRACT

Methods for diagnosing and treating conditions associated with life-threatening neurological complications are provided. The methods involve in some aspects the identification of oxLDL and LOX-1 as critical players in pregnant subjects and in some cases subjects having severe preeclampsia (early onset preeclampsia). Related products and kits are also provided.

RELATED APPLICATIONS

This application is a divisional application which claims the benefitunder 35 U.S.C. § 120 of U.S. application Ser. No. 14/045,038, entitled“OXIDIZED LDL AS A BIOMARKER FOR NEUROLOGICAL COMPLICATIONS OFPREGNANCY” filed on Oct. 3, 2013, which claims priority under 35 U.S.C.§ 119(e) to U.S. Provisional Application Ser. No. 61/709,297, entitled“OXIDIZED LDL AS A BIOMARKER FOR NEUROLOGICAL COMPLICATIONS OFPREGNANCY,” filed on Oct. 3, 2012, which are herein incorporated byreference in their entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. NS045940and NS045940-0651 awarded by NINDS. The government has certain rights inthis invention.

FIELD OF THE INVENTION

Aspects of the invention relate to diagnosing and predictingneurological complications of pregnancy and/or preeclampsia as well asrelated products and kits. Methods of treating preeclampsia are alsoprovided.

BACKGROUND

Preeclampsia is a condition that develops during the last half ofpregnancy and is associated with significant maternal and fetalmorbidity and mortality. Because there is no effective screening test todiagnose or assess the risk of developing preeclampsia and associatedhypertensive disorders, pregnant women cannot receive effectivemonitoring or treatment until long after complications associated withthe disorders, including increased blood pressure and proteinuria, havedeveloped. In addition, pregnant women with little to no risk ofdeveloping preeclampsia or associated hypertensive disorders mustundergo unnecessary testing for symptoms throughout their pregnancybecause there is no effective means by which caregivers may exclude themfrom risk in the early stages of pregnancy. Additionally, patientshaving milder forms of preeclampsia are not distinguished from thosepatients having severe preeclampsia and thus may receive unnecessarytreatments.

SUMMARY OF INVENTION

In some aspects the invention is a method for identifying a subject atrisk of neurological complications associated with pregnancy byisolating a tissue sample from a pregnant subject, determining a levelof oxLDL in the pregnant subject, determining the subject is at risk ofneurological complications associated with pregnancy if the oxLDL levelsare greater than a control level. The oxLDL levels in some embodimentsare greater than 1,600 ng/ml.

The tissue sample is a blood sample in some embodiments.

In some embodiments the method also involves providing a course oftreatment for the subject if the subject is identified as at risk ofneurological complications associated with pregnancy. Optionally thesubject may be administered an anti-seizure prophylaxis, such asmagnesium sulfate.

The levels of oxLDL may be measured using any known methods in the art.For instance, the levels of oxLDL may be measured using an antibodyassay and/or using a kit for detecting oxLDL.

The subject, in some embodiments is in the first, second or thirdtrimester of pregnancy.

The method may also involve measuring the blood pressure and/orcholesterol of the subject. In some instances the blood pressure of thesubject is within normal levels and/or the cholesterol of the subject isabove normal levels.

A method for treating a subject is also provided according to aspects ofthe invention. The method involves administering to a pregnant subject,optionally having preeclampsia, wherein the pregnancy is associated withneurological complications, an oxLDL inhibitor or LOX-1 inhibitor in aneffective amount to treat the subject. In some embodiments the pregnantsubject is identified as a subject having elevated levels of oxLDL.

In some embodiments the subject does not have an inflammatory disorderand/or the subject has not been diagnosed with an inflammatory disorder.

The subject is administered an oxLDL inhibitor in some embodiments. TheoxLDL inhibitor may be an anti-oxLDL binding peptide such as ananti-oxLDL antibody, for instance.

In other embodiments the subject is administered a LOX-1 inhibitor. TheLOX-1 inhibitor may be, for example, an anti-LOX-1 binding peptide, suchas an anti-LOX-1 antibody or an inhibitory nucleic acid.

The invention in other aspects is a method for treating a subject byadministering to a pregnant subject having a condition associated withneurological complications an antioxidant in an effective amount totreat the subject. In some embodiments the subject has been diagnosedwith preeclampsia associated with neurological complications accordingto the methods described herein. In other embodiments the antioxidant isa superoxide dismutase mimetic, Tempol, ONOO⁻ scavengers such as FeTMPyP(Fe(III)tetrakis (1-methyl-4-pyridyl) porphyrin pentachlorideporphyrinpentachloride) and FeTPPS(5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato Iron (III),Chloride), a compound that reduces reactive nitrogen species, a compoundthat reduces reactive oxygen species, and ebselen.

In other aspects of the invention, a kit is provided. The kit includesone or more containers housing a reagent for detecting oxLDL levels andinstructions for diagnosing pregnancy associated with neurologicalcomplications. In some embodiments the reagent for detecting oxLDL is anoxLDL antibody. In other embodiments the reagent for detecting oxLDL isan oxLDL nucleic acid. In yet other embodiments the instructions fordiagnosing pregnancy associated with neurological complications refer todetermining a level of oxLDL in a pregnant subject wherein the subjectis at risk of neurological complications associated with pregnancy ifthe oxLDL levels are greater than a control level.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a flowchart showing the proposed mechanism leading toneurological complications in early-onset preeclampsia. Increased levelsof oxLDL in plasma from EPE women bind to its receptor LOX-1 and induceproduction of superoxide (O₂ ⁻) that will generate peroxynitrite(ONOO⁻). Increased generation of ONOO⁻ causes disruption of BBB andincreased BBB permeability that is responsible for neurologicalcomplications in preeclampsia.

FIG. 2A-2D are sets of graphs depicting the effect of plasma fromnonpregnant (NonP), normal pregnant (NP), late-onset preeclamptic (LPE)and early onset (EPE) women on blood brain barrier (BBB) permeability.Graphs show: (A) intravascular pressure drop, (B) volume flux, (C)intravascular filtration (Jv/s), and (D) hydraulic conductivity (L_(p))as a measure of BBB permeability of cerebral veins of NonP rats inresponse to plasma from EPE, LPE, NP and NonP women. Plasma from EPE andLPE women significantly increased BBB permeability compared to NonPwomen. Plasma from EPE women significantly increased BBB permeabilitycompared to all other groups, including LPE women. (

P<0.01 vs. NonP; **P<0.01 vs. all; NonP=nonpregnant; NP=normal pregnant;LPE=late-onset preeclampsia; EPE=early-onset preeclampsia)

FIG. 3A-3C are sets of graphs demonstrating the presence of oxLDL in EPEand its effect on BBB permeability. FIG. 3A shows a graph of OxLDLlevels in plasma from women with EPE compared to LPE, NP and NonP women.Plasma from EPE women had a 260% increase in oxLDL compared to plasmafrom LPE women. FIG. 3B shows a graph of hydraulic conductivity (L_(p))at 36 minutes as a measure of BBB permeability in cerebral veins fromNonP rats in response to plasma from LPE women and plasma from EPE womenwith or without the addition of 10 μg/ml LOX-1 antibody. The LOX-1antibody inhibited the increased BBB permeability induced by plasma fromEPE women. FIG. 3C shows a graph of L_(p) as a measure of BBBpermeability in cerebral veins from NonP rats in response to plasma fromNonP and EPE women and plasma from NonP women with addition of 3.5 μg/mlexogenous oxLDL. Exogenous oxLDL significantly increased BBBpermeability to the same levels as plasma from EPE women. (**P<0.01 vs.all; NonP=nonpregnant; NP=normal pregnant; LPE=late-onset preeclampsia;EPE=early-onset preeclampsia).

FIGS. 4A and 4B are sets of graphs showing the effect of theperoxynitrite scavenger FeTMPyP on BBB permeability in EPE induced byoxLDL. FIG. 4A is a graph showing hydraulic conductivity (L_(P)) at 36minutes as a measure of BBB permeability in cerebral veins of NonP ratsin response to plasma from LPE and plasma from EPE women with andwithout the addition of 50 μM FeTMPyP. FeTMPyP inhibited the BBBpermeability induced by plasma from EPE women. FIG. 4B is a graphshowing L_(p) as a measure of BBB permeability in response to untreatedof plasma from NonP and EPE women, and in response to plasma from NonPwomen plus 3.5 μg/ml oxLDL and 50 μM FeTMPyP. FeTMPyP inhibited the BBBpermeability induced by 3.5 μg/ml exogenous oxLDL in plasma from NonPwomen. (**P<0.01 vs. all; LPE=late-onset preeclampsia; EPE=early-onsetpreeclampsia).

FIGS. 5A and 5B are sets of graphs showing the effects of pathologicallyhigh levels of cholesterol in pregnant rats. FIG. 5A shows hydraulicconductivity (L_(p)) as a measure of BBB permeability at 36 minutes incerebral veins from late-pregnant control (LP-CTL) rats in response toLP-CTL plasma and from late-pregnant high cholesterol treated (LP-HC)rats in response to LP-HC plasma with and without the addition of 10μg/ml LOX-1 antibody. LP-HC rats showed a significant increase in BBBpermeability that could be inhibited with the addition of LOX-1antibody. FIG. 5B shows mRNA expression of LOX-1 in cerebral veins fromLP-CTL and LP-HC animals. There was no difference in mRNA expression ofLOX-1 in cerebral veins from LP-HC vs. LP-CL animals. (*P<0.05 vs. all;LP-CTL=late pregnant control; LP-HC=late pregnant high cholesteroltreated).

DETAILED DESCRIPTION OF INVENTION

Vasogenic brain edema is a major contributor to the occurrence ofneurological symptoms in preeclampsia and is the result of increasedcerebrovascular permeability and disruption of the blood-brain barrier(BBB). The cerebral endothelium that comprises the BBB contains complexhigh electrical resistance tight junctions that prevent traffic of ionsand large proteins into the brain, resulting in low hydraulicconductivity (L_(p)). BBB disruption causes vasogenic edema and thepassage of damaging proteins and plasma constituents into the brainparenchyma. The inventor has previously established that circulatingfactors in plasma from preeclamptic, but not normal pregnant women,increased BBB permeability, suggesting involvement of circulatingfactors in BBB disruption in preeclampsia. In that study, plasma frompreeclamptic women with severe disease (defined with American College ofObstetricians and Gynecologists criteria) was used, but early-onsetpreeclampsia (EPE) and late-onset preeclampsia (LPE) were notdistinguished. The invention is based at least in part on the discoverythat the plasma from EPE women has a greater impact on BBB disruptionthat underlies neurological symptoms and further involves the discoveryof a key factor involved in that process. The increased BBB permeabilityin response to the plasma from the preeclamptic women was prevented bythe inhibition of vascular endothelial growth factor (VEGF) signaling.However, VEGF levels were not increased in plasma from preeclampticwomen, suggesting other circulating factors are responsible forincreasing BBB permeability. Understanding what circulating factors inplasma from preeclamptic women increase BBB permeability and itsunderlying mechanism may provide a therapeutic target to prevent and/orpredict neurological complications during preeclampsia.

It has been shown that increased oxidized LDL (oxLDL) levels andexpression of LOX-1 (oxLDL receptor) in the systemic vasculature inwomen with preeclampsia. Here we discovered that oxLDL binding to LOX-1plays a critical role in BBB disruption that underlies the pathogenesisof neurological complications of preeclampsia. Specifically it has beenshown according to the invention that increased oxLDL in EPE, throughactivation of LOX-1, is an underlying mechanism by which BBB disruptionoccurs in EPE women. The plasma from EPE women significantly increasedBBB permeability compared to LPE women through increased levels ofoxLDL, activation of LOX-1 and subsequent ONOO⁻ generation.Additionally, an animal model of high-cholesterol in pregnancy wasdeveloped to show that elevated LDL adversely affects pregnancy outcomeand causes BBB disruption as seen in EPE.

It was also demonstrated, according to the invention, as shown in theExamples, that circulating factors in plasma from EPE womensignificantly increased BBB permeability compared to plasma from LPEwomen that was prevented by inhibiting LOX-1. Circulating oxLDL, themajor ligand of LOX-1, was significantly increased in plasma from EPEwomen (by 260%) compared to LPE women. Exogenous oxLDL added to plasmafrom non-pregnant women also increased BBB permeability comparable toEPE. Additionally, the selective ONOO⁻ decomposition catalyst FeTMPyPinhibited increased BBB permeability induced by plasma from EPE women orinduced by exogenous oxLDL. Finally, a rat model of pathologically highlipid levels in pregnancy showed a significant increase in bloodpressure and adverse effects on pregnancy outcome, similar to EPE. BBBpermeability was also increased in response to high levels ofcholesterol that could be prevented by blocking LOX-1. Taken together,these results show for the first time that plasma from EPE womensignificantly increase BBB permeability compared to LPE women throughincreased oxLDL levels that activate LOX-1, resulting in increased ONOO⁻generation.

Therefore, the invention involves, in some aspects, a method foridentifying a subject at risk of neurological complications associatedwith pregnancy. The method is performed by isolating a tissue samplefrom a pregnant subject, determining a level of oxLDL in the pregnantsubject, and determining that the subject is at risk of neurologicalcomplications associated with pregnancy if the oxLDL levels are greaterthan a control level.

A “subject at risk of neurological complications associated withpregnancy”, as used herein, is a subject that is pregnant that coulddevelop neurological complications or in some instances a subject thathas recently pregnant and experienced neurological complications such asseizure but is no longer pregnant. In some instances the pregnantsubject has been diagnosed with preeclampsia.

As used herein, “preeclampsia” is a disorder that occurs during, whichaffects both the mother and the unborn baby and is associated withhypertension and proteinuria after the 20^(th) week of pregnancy.Patients having preeclampsia can be classified as EPE and LPE.Clinically preeclampsia is defined according to well-establishedcriteria, such as a blood pressure of at least 140/90 mm Hg and urinaryexcretion of at least 0.3 grams of protein in a 24-hour urinary proteinexcretion (or at least +1 or greater on dipstick testing), each on twooccasions 4-6 hours apart. EPE is also sometimes referred to as, “severepreeclampsia” and can be defined clinically, as a blood pressure of atleast 160/110 mm Hg on at least 2 occasions 6 hours apart and greaterthan 5 grams of protein in a 24-hour urinary protein excretion orpersistent +3 proteinuria on dipstick testing. Severe preeclampsia mayinclude HELLP syndrome (hemolysis, elevated liver enzymes, low plateletcount). Other elements of EPE may include in-utero growth restriction(IUGR) in less than the 10% percentile according to the US demographics,persistent neurologic symptoms (headache, visual disturbances),epigastric pain, oliguria (less than 500 mL/24 h), serum creatininegreater than 1.0 mg/dL, elevated liver enzymes (greater than two timesnormal), thrombocytopenia (<100,000 cells/μL) and neurologicalcomplications.

Neurological complications associated with pregnancy include but are notlimited to seizures, coma, focal motor deficits, cortical blindness, andcerebrovascular hemorrhage. Neurological symptoms are the most seriousand life-threatening complications of preeclampsia (1). The appearanceof neurological complications accounts for at least 75% of maternalmortality worldwide (2, 3). However, neurological complications do notoccur in all women diagnosed with preeclampsia, suggesting differencesin the pathogenesis of this disease. Epidemiologic studies have shownthat neurological complications occur most often in early-onsetpreeclampsia (EPE) where hypertension and proteinuria occur before 34weeks of gestation, compared to late-onset preeclampsia (LPE) thatdevelops after 34 weeks of gestation (4-7). These findings importantlysuggest that EPE is a form of preeclampsia that affects the brain moreseverely, contributing to neurological complications.

The methods involve isolation of a tissue sample from a pregnantsubject. A pregnant subject, as used herein is a female mammaliansubject that has been identified as being pregnant or who is at risk ofbeing pregnant. A subject is at risk of being pregnant if the subject iscapable of having a child and who has been exposed to sperm. Inpreferred embodiments the pregnant subject is a human subject. A controlsubject is a normal subject who has been determined not to be pregnant,either by inability to become pregnant or lack of exposure to spermduring critical time points. The isolated tissue sample is a tissue inwhich oxLDL is expressed. An exemplary tissue sample is blood or aportion thereof, such as plasma.

The pregnant subject may be in any stage of the pregnancy. For instance,the subject may be in the first, second or third trimester of pregnancy.

The subject may be diagnosed as having preeclampsia or such diagnosismay be revealed through the testing methods of the invention. Diagnosiscan be achieved using known methods in the art. For instance, the bloodpressure of the subject may be measured and may be elevated. However,elevated blood pressure is not essential for a subject to havepreeclampsia. Therefore the blood pressure of the subject may be withinnormal levels. The subject may also be subjected to a test measuringcholesterol levels. In some embodiments the cholesterol levels of thesubject are above normal levels.

A level of oxLDL in the pregnant subject is determined in order toassess the status of the preeclampsia. The levels of oxLDL serve as amarker indicating the presence or absence of neurological complicationsassociated with preeclampsia. The term “marker” refers to an organicbiomolecule, preferably, a lipid that is differentially present in asample taken from a subject having EPE as compared to a comparablesample taken from a subject who has LPE or from a normal subject, whodoes not have preeclampsia and/or is not pregnant. A marker isdifferentially present in samples from subjects having EPE, if it ispresent at an elevated level in the subject with EPE, as compared to acontrol level. A control level may be a level of oxLDL found in samplesfrom normal subjects and/or subjects having LPE or alternatively astandard control level. A subject having elevated levels of oxLDL is onewho is at risk of neurological complications associated with pregnancy.

OxLDL has a greater negative charge compared to native LDL and thusoxLDL, but not native LDL, is available to bind the lectin-like oxidizedLDL receptor 1 (LOX-1) that is predominantly expressed on endothelialcells. Under normal physiological conditions LOX-1 activity is low, butits increased activation during disease states such as atherosclerosis,diabetes and hypertension causes endothelial dysfunction. LOX-1activation rapidly stimulates the production of superoxide inendothelial cells, mainly through activation of NADPH oxidase.Superoxide decreases the concentration of nitric oxide (NO) by bindingNO to form peroxynitrite (ONOO⁻), a relatively stable reactive oxygenand nitrogen species that has deleterious effects on cell viability andendothelial function. ONOO⁻ generation has been reported in the systemicvasculature in women with preeclampsia, however, whether ONOO⁻generation, secondary to LOX-1 activation, is also involved indisrupting the BBB during preeclampsia is not known. It has beendemonstrated according to the invention that ONOO⁻ generation, secondaryto LOX-1 activation, causes increased BBB permeability in women withEPE.

Once a subject is diagnosed as having increased oxLDL levels duringpregnancy a therapeutic course of treatment can be applied. The subjectbeing treated is a subject having pregnancy associated with neurologicalcomplications. The subject may be identified as a subject havingelevated levels of oxLDL. Alternatively, the subjects having pregnancyassociated with neurological complications treated according to themethods of the invention may be identified by other methods known in theart. For instance the subject may be a pregnant subject who hasexperienced seizures. In some embodiments the subject does not have aninflammatory disorder. In other embodiments the subject has not beendiagnosed with an inflammatory disorder.

The therapeutic treatment applied to the subject may be a traditionaltherapeutic for treating the symptoms of preeclampsia. For instance, thesubject may be treated with an anti-seizure prophylaxis or therapeutic.The anti-seizure prophylaxis may be, for instance, magnesium sulfate.

Alternatively, the therapeutic method may be a therapeutic method of theinvention. For instance, the therapeutic method may involveadministration of an oxLDL inhibitor, an LOX-1 inhibitor, or anantioxidant.

An oxLDL inhibitor, as used herein, refers to a compound that can reduceoxLDL activity and/or levels in a subject. These inhibitors include butare not limited to small molecule inhibitors, nucleic acid inhibitorsand peptide based inhibitors. Small molecule inhibitors include but arenot limited to atorvastatin, as well as analogs and variants thereof. ALOX-1 inhibitor, as used herein, refers to a compound that can reduceLOX-1 activity and/or levels in a subject. These inhibitors include butare not limited to small molecule inhibitors, nucleic acid inhibitorsand peptide based inhibitors. Small molecule inhibitors include but arenot limited to statins and procyanidins, as well as analogs and variantsthereof.

The oxLDL inhibitor and LOX-1 inhibitor may also be binding peptides. Ananti-oxLDL binding peptide is a peptide that binds specifically to oxLDLand interferes with its activity. The binding peptide may be ananti-oxLDL antibody. An anti-LOX-1 binding peptide is a peptide thatbinds specifically to LOX-1 and interferes with its activity. Thebinding peptide may be an anti-LOX-1 antibody. Antibodies, includingfragments thereof, single chain antibodies etc. are well known in theart.

The subject may also be administered an antioxidant in an effectiveamount to treat the subject. An antioxidant is a compound that inhibitsthe oxidation of other molecules. Antioxidants include but are notlimited to superoxide dismutase mimetics, Tempol, ONOO⁻ scavengers suchas FeTMPyP and FeTPPS, and ebselen.

When used in combination with the therapies of the invention the dosagesof known therapies may be reduced in some instances, to avoid sideeffects.

The active agents of the invention are administered to the subject in aneffective amount for treating the subject. An “effective amount”, forinstance, is an amount necessary or sufficient to realize a desiredbiologic effect. For instance an effective amount is that amountsufficient to prevent or inhibit neurological complications ofpreeclampsia.

The effective amount of a compound of the invention in the treatment ofa subject may vary depending upon the specific compound used, the modeof delivery of the compound, and whether it is used alone or incombination. The effective amount for any particular application canalso vary depending on such factors as the type and/or degree ofinfection in a subject, the particular compound being administered fortreatment, the size of the subject, or the severity of the disorder. Oneof ordinary skill in the art can empirically determine the effectiveamount of a particular molecule of the invention without necessitatingundue experimentation. Combined with the teachings provided herein, bychoosing among the various active compounds and weighing factors such aspotency, relative bioavailability, patient body weight, severity ofadverse side-effects and preferred mode of administration, an effectiveprophylactic or therapeutic treatment regimen can be planned which doesnot cause substantial toxicity in and of itself and yet is entirelyeffective to treat the particular subject.

Toxicity and efficacy of the protocols of the present invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Prophylactic and/or therapeutic agents that exhibit largetherapeutic indices are preferred. While prophylactic and/or therapeuticagents that exhibit toxic side effects may be used, care should be takento design a delivery system that targets such agents to the site ofaffected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays, animal studies and humanstudies can be used in formulating a range of dosage of the prophylacticand/or therapeutic agents for use in humans. The dosage of such agentslies preferably within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any agent used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

As used herein, the term treat, treated, or treating when used withrespect to a disorder refers to a prophylactic treatment which increasesthe resistance of a subject to development of the disease or, in otherwords, decreases the likelihood that the subject will develop thedisease as well as a treatment after the subject has developed thedisease in order to fight the disease, prevent the disease from becomingworse, or slow the progression of the disease compared to in the absenceof the therapy. A prophylactic is desired for instance if a pregnantsubject has had prior instances of EPE in prior pregnancies.

Multiple doses of the molecules of the invention are also contemplated.In some instances, when the molecules of the invention are administeredwith another therapeutic, for instance, a chemotherapeutic agent asub-therapeutic dosage of either or both of the molecules may be used. A“sub-therapeutic dose” as used herein refers to a dosage which is lessthan that dosage which would produce a therapeutic result in the subjectif administered in the absence of the other agent.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents, dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. Moreover, for animal (e.g., human) administration, itwill be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards. The compounds are generally suitable foradministration to humans. This term requires that a compound orcomposition be nontoxic and sufficiently pure so that no furthermanipulation of the compound or composition is needed prior toadministration to humans.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences(1990), incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated. Thecompounds may be sterile or non-sterile.

The agent may comprise different types of carriers depending on whetherit is to be administered in solid, liquid or aerosol form, and whetherit need to be sterile for such routes of administration as injection.The present invention can be administered intravenously, intradermally,intraarterially, intralesionally, intratumorally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g., aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in creams, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences (1990), incorporated herein byreference).

In any case, the composition of oxLDL or LOX-1 inhibitor may furthercomprise various antioxidants to retard oxidation of one or morecomponents as well as to treat the disease. Additionally, the preventionof the action of microorganisms can be brought about by preservativessuch as various antibacterial and antifungal agents, including but notlimited to parabens (e.g., methylparabens, propylparabens),chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The agent may be formulated into a composition in a free base, neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups also can be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

The compounds of the invention may be administered directly to a tissue.Direct tissue administration may be achieved by direct injection. Thecompounds may be administered once, or alternatively they may beadministered in a plurality of administrations. If administered multipletimes, the compounds may be administered via different routes. Forexample, the first (or the first few) administrations may be madedirectly into the affected tissue while later administrations may besystemic.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients. In general, a pharmaceutical composition comprises thecompound of the invention and a pharmaceutically-acceptable carrier.Pharmaceutically-acceptable carriers for nucleic acids, small molecules,peptides, monoclonal antibodies, and antibody fragments are well-knownto those of ordinary skill in the art. As used herein, apharmaceutically-acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients.

Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other materials which arewell-known in the art. Exemplary pharmaceutically acceptable carriersfor peptides in particular are described in U.S. Pat. No. 5,211,657.Such preparations may routinely contain salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The compounds of the invention may be formulated into preparations insolid, semi-solid, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, depositories, inhalants andinjections, and usual ways for oral, parenteral or surgicaladministration. The invention also embraces pharmaceutical compositionswhich are formulated for local administration, such as by implants.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids, such as a syrup,an elixir or an emulsion.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. Techniques forpreparing aerosol delivery systems are well known to those of skill inthe art. Generally, such systems should utilize components which willnot significantly impair the biological properties of the active agent(see, for example, Sciarra and Cutie, “Aerosols,” in Remington'sPharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporatedby reference). Those of skill in the art can readily determine thevarious parameters and conditions for producing aerosols without resortto undue experimentation.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the agents of the invention to the subject. Biodegradablematrices are preferred. Such polymers may be natural or syntheticpolymers. Synthetic polymers are preferred. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

The invention also encompasses various assays for diagnostic andtherapeutic purposes as discussed above and related kits to achieve themethods. The diagnostic methods may achieve for instance using proteinor lipoprotein detection assays for detecting and measuring expressionlevels of oxLDL.

Detection of a protein or lipoprotein in a test sample involves routinemethods. The skilled artisan can detect the presence or absence of aprotein using well known methods. One such method is an immunoassay. Ingeneral, immunoassays involve the binding of proteins in a sample to asolid phase support such as a plastic surface. Detectable antibodies arethen added which selectively bind to the protein of interest. Detectionof the antibody indicates the presence of the protein. The detectableantibody may be a labeled or an unlabeled antibody. Unlabeled antibodymay be detected using a second, labeled antibody that specifically bindsto the first antibody or a second, unlabeled antibody which can bedetected using labeled protein A, a protein that complexes withantibodies. Various immunoassay procedures are described in Immunoassaysfor the 80's, A. Voller et al., Eds., University Park, 1981, and TheImmunoassay Handbook, Third Edition, David Geoffrey Wild (Ed), 2005which are incorporated herein by reference.

Simple immunoassays such as a dot blot and a Western blot involve theuse of a solid phase support which is contacted with a test sample. Anyproteins present in the test sample bind the solid phase support and canbe detected by a specific, detectable antibody preparation. Theintensity of the signal can be measured to obtain a quantitativereadout. Other more complex immunoassays include forward assays for thedetection of a protein in which a first anti-protein antibody bound to asolid phase support is contacted with the test sample. After a suitableincubation period, the solid phase support is washed to remove unboundprotein. A second, distinct anti-protein antibody is then added which isspecific for a portion of the specific protein not recognized by thefirst antibody. The second antibody is preferably detectable. After asecond incubation period to permit the detectable antibody to complexwith the specific protein bound to the solid phase support through thefirst antibody, the solid phase support is washed a second time toremove the unbound detectable antibody. Alternatively, in a forwardsandwich assay a third detectable antibody, which binds the secondantibody is added to the system. Other types of immunometric assaysinclude simultaneous and reverse assays. A simultaneous assay involves asingle incubation step wherein the first antibody bound to the solidphase support, the second, detectable antibody and the test sample areadded at the same time. After the incubation is completed, the solidphase support is washed to remove unbound proteins. The presence ofdetectable antibody associated with the solid support is then determinedas it would be in a conventional assays. A reverse assay involves thestepwise addition of a solution of detectable antibody to the testsample followed by an incubation period and the addition of antibodybound to a solid phase support after an additional incubation period.The solid phase support is washed in conventional fashion to removeunbound protein/antibody complexes and unreacted detectable antibody.

A number of methods are well known for the detection of antibodies. Forinstance, antibodies can be detectably labeled by linking the antibodiesto an enzyme and subsequently using the antibodies in an enzymeimmunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), such asa capture ELISA. The enzyme, when subsequently exposed to its substrate,reacts with the substrate and generates a chemical moiety which can bedetected, for example, by spectrophotometric, fluorometric or visualmeans. Enzymes which can be used to detectably label antibodies include,but are not limited to malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

A detectable label is a moiety, the presence of which can be ascertaineddirectly or indirectly. Generally, detection of the label involves anemission of energy by the label. The label can be detected directly byits ability to emit and/or absorb photons or other atomic particles of aparticular wavelength (e.g., radioactivity, luminescence, optical orelectron density, etc.). A label can be detected indirectly by itsability to bind, recruit and, in some cases, cleave another moiety whichitself may emit or absorb light of a particular wavelength (e.g.,epitope tag such as the FLAG epitope, enzyme tag such as horseradishperoxidase, etc.). An example of indirect detection is the use of afirst enzyme label which cleaves a substrate into visible products. Thelabel may be of a chemical, peptide or nucleic acid molecule naturealthough it is not so limited. Other detectable labels includeradioactive isotopes such as P32 or H3, luminescent markers such asfluorochromes, optical or electron density markers, etc., or epitopetags such as the FLAG epitope or the HA epitope, biotin, avidin, andenzyme tags such as horseradish peroxidase, *-galactosidase, etc. Thelabel may be bound to a peptide during or following its synthesis. Thereare many different labels and methods of labeling known to those ofordinary skill in the art. Examples of the types of labels that can beused in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbioluminescent compounds. The coupling or conjugation of these labels tothe antibodies can be performed using standard techniques common tothose of ordinary skill in the art.

Another labeling technique which may result in greater sensitivityconsists of coupling the molecules described herein to low molecularweight haptens. These haptens can then be specifically altered by meansof a second reaction. For example, it is common to use haptens such asbiotin, which reacts with avidin, or dinitrophenol, pyridoxal, orfluorescein, which can react with specific anti-hapten antibodies.

In one embodiment, a kit comprises an inhibitor of oxLDL and/or LOX-1and instructions for administering the same. The kit may furthercomprise devices for administering the inhibitors, and/or othertherapeutics or diagnostics related to the therapy. The kit may alsoinclude antioxidants or other useful therapeutics as well as articles toenable delivery of the compounds to the subject.

In other embodiments the kit may include a reagent for detecting oxLDLlevels and instructions for diagnosing pregnancy associated withneurological complications. Reagents for detecting oxLDL include bindingpeptides such as oxLDL antibodies or oxLDL nucleic acid.

The instructions for diagnosing pregnancy associated with neurologicalcomplications may refer to determining a level of oxLDL in a pregnantsubject. “Instructions” can define a component of promotion, andtypically involve written instructions on or associated with packagingof compositions of the invention. Instructions also can include any oralor electronic instructions provided in any manner.

Thus the agents described herein may, in some embodiments, be assembledinto pharmaceutical or diagnostic or research kits to facilitate theiruse in therapeutic, diagnostic or research applications. A kit mayinclude one or more containers housing the components of the inventionand instructions for use. Specifically, such kits may include one ormore agents described herein, along with instructions describing theintended therapeutic application and the proper administration of theseagents. In certain embodiments agents in a kit may be in apharmaceutical formulation and dosage suitable for a particularapplication and for a method of administration of the agents.

The kit may be designed to facilitate use of the methods describedherein by physicians and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for human administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19. Pharmaceutically acceptablesalts of the compounds of this invention include those derived fromsuitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

As used herein, the term “prodrug” means a biologically activederivative of a compound that can hydrolyze, oxidize, or otherwise reactunder biological conditions (in vitro or in vivo) to provide thepharmacologically active compound. In this instance, the “prodrug” is acompound administered to a subject, and the pharmacologically activecompound is the “active metabolite thereof.” In certain cases, a prodrughas improved physical and/or delivery properties over the parentcompound. Prodrugs are typically designed to enhance pharmaceuticallyand/or pharmacokinetically based properties associated with the parentcompound. The advantage of a prodrug can lie in its physical properties,such as enhanced water solubility for parenteral administration atphysiological pH compared to the parent compound, or it enhancesabsorption from the digestive tract, or it may enhance drug stabilityfor long-term storage.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

Summary:

The following examples demonstrate for the first time that high levelsof oxLDL in EPE increase BBB permeability through LOX-1 activation andsubsequent ONOO⁻ generation. Also, a rat model of high cholesterol inpregnancy confirmed the damaging effects of high levels of LDL that wascomparable to EPE. This is the first report that identifies that onlycirculating factors in EPE increase BBB permeability compared to LPE. Inaddition, the data establishes a mechanism for BBB disruption in EPEthat might be helpful in improving the identification and treatment ofneurological complications in pregnant women. Further studies arerequired to determine how this BBB disruption induced by oxLDL leads toactual neurological symptoms, which could lead to treatment orprevention of these complications in preeclampsia.

The cerebral endothelium that comprises the BBB provides a strongprotective mechanism against vasogenic brain edema (11). As we haveshown previously, circulating factors released in preeclampsia candisrupt the BBB that may lead to vasogenic edema, the greatestcontributor of neurological complications in preeclampsia (13). Here,the inventors have demonstrated for the first time that intraluminalexposure of plasma from EPE women in the cerebral vein of Galen for 3hours significantly increased BBB permeability compared to plasma fromLPE or NP women. These new findings are of great clinical importance asthey demonstrate that EPE women are at significant risk for neurologicalcomplications and thereby confirm the epidemiologic studies. Priorstudies have not differentiated effects of LPE vs. EPE on BBBpermeability. Neal et al. found increased permeability in systemicmesenteric arteries from frogs in response to plasma from severecompared to mild preeclamptic women, however, the severity ofpreeclampsia was strongly reflected in time of onset (32). We show thatEPE and LPE have different etiologies, arising from greater placentalunderperfusion and subsequent release of circulating factors in EPEcompared to LPE and that EPE and LPE should be considered as twoentities displaying at least partially different etiologies with EPEhaving the greatest risk for neurological complications.

We found that oxLDL levels were increased in plasma from EPE women butnot in plasma from LPE women that was comparable to NP women. Thisincrease in EPE only compared to LPE and NP may be related to the factthat oxidative stress was found only in placentas from EPE women,suggesting greater capacity for oxidative modification of LDL in EPE.Since oxLDL was increased in plasma from EPE women only, this importantfinding provides an important biomarker for neurological complicationsin preeclamptic women.

OxLDL mainly acts through its receptor LOX-1, which has been studiedextensively in pathological states such as atherosclerosis, coronaryarterial heart disease and hypertension (19, 20). However, dataregarding LOX-1 activation in preeclampsia are scarce. LOX-1 wasassociated with preeclampsia for the first time in a study in 2005 thatshowed upregulation of LOX-1 in preeclamptic hypoxic placentas (42).Recently, it was found that upregulation of LOX-1 in HUVECs occurred inresponse to plasma from preeclamptic women and in a rat model ofpreeclampsia (22, 23). Here, we show for the first time involvement ofLOX-1 in increasing BBB permeability in plasma from EPE women. Also, weshowed that exogenous oxLDL to the levels as measured in plasma from EPEwomen significantly increased BBB permeability in cerebral veins,comparable to EPE women. Thus, increased oxLDL may not be just abiomarker for neurological symptoms in EPE, but also the underlyingcause. We propose a novel mechanism in that high levels of circulatingoxLDL increase BBB permeability in EPE through LOX-1 activation. To ourknowledge, only few studies investigated oxLDL and vascularpermeability. One study using mouse cultured cerebral endothelial cellsshowed that oxLDL is able to increase cerebral permeability (30). In thesystemic vasculature, Nakano et al. showed increased vascularpermeability in mesenteric arteries in spontaneous hypertensive ratspretreated with oxLDL that was also inhibited by a LOX-1 antibody (43).Further, for this study, we did not determine possible upregulation ofLOX-1 after exposure to plasma from EPE women because cerebral veinswere only incubated for 3 hours. Sankaralingam et al. found LOX-1upregulation in HUVECs in response to plasma from preeclamptic womenafter 24 hrs, while after 6 hrs oxidative stress was already increasedwithout upregulation of LOX-1, confirming that increased ligands such asoxLDL activating LOX-1 already have damaging effects on the endothelium.It remains to be determined in cultured cerebral endothelial cells iflonger incubation would cause upregulation of LOX-1 expression inresponse to plasma from EPE women as seen in HUVECs in response topreeclamptic plasma (23).

LOX-1 activation has been shown to induce several intracellularsignaling pathways, such as increased expression of chemokines andadhesion molecule, triggering of the CD40/CD40L pathway that activatesthe inflammatory cascade and increased production of reactive oxygenspecies in endothelial cells (44). Here, we show that BBB permeabilityinduced by either plasma from EPE women or exogenous oxLDL to the levelsof EPE women was inhibited with FeTMPyP, demonstrating that ONOO⁻generation caused the increased BBB permeability induced by oxLDL/LOX-1activation. ONOO⁻ is generated by the reactive oxygen species superoxideand NO and is known to have deleterious effects on endothelial function(18, 27). Several studies confirm our findings that oxLDL/LOX-1activation induces ROS and ONOO⁻ generation. Cominacini et al. showed inbovine artery cultured cells that binding of oxLDL to LOX-1 decreasedthe intracellular concentration of NO by inducing the production ofsuperoxide through NADPH oxidase (25). Another study using HUVECs foundthat ONOO⁻ generation was stimulated by oxLDL (45). One studyinvestigating preeclampsia, showed increased activity of NADPH oxidaseand ONOO⁻ generation in HUVECs after exposure to plasma frompreeclamptic women (23). Because NADPH oxidase expression and activityis greater in the cerebral vessels compared to systemic vasculature(46), the brain may be especially vulnerable for activation of NADPHoxidases and subsequent ONOO⁻ generation. Importantly, ONOO⁻ alsostimulates LOX-1 and a self-perpetuating mechanism develops that couldcause extensive endothelial damage with deleterious effects for thematernal cerebral vasculature (18). In addition, these findings wouldexplain the results from our previous study in which the increased BBBpermeability was prevented by VEGF receptor inhibition (13).Phosphorylation of VEGF receptors results in release of NO (47), thus,its inhibition would decrease the level of NO available for ONOO⁻generation, preventing BBB disruption (FIG. 1).

Finally, we created a rat model with pathologically high levels of LDLin pregnancy to examine pregnancy outcome, blood pressure and BBBpermeability with possible LOX-1 involvement as seen in EPE. LP-HC ratsshowed a modest but significant increase in blood pressure and a lowerbirth weight, as seen in EPE, compared to LP-CTL animals. Interestingly,these animals also show significantly increased BBB permeability thatcould be inhibited by blocking LOX-1, suggesting oxidative modificationof high levels of LDL in the LP-HC rats comparable as seen in EPE. Incontrast to other studies, we did not find upregulation of LOX-1 in thismodel of preeclampsia. A possible explanation for this difference couldbe that other studies used HUVECs or large conduct arteries such as themesenteric artery and aorta, whereas we examined the effect of highcholesterol on cerebral arteries. Regardless, increased BBB permeabilityin response to increased oxLDL can occur without upregulation of LOX-1expression, as seen in EPE women.

Materials and Methods:

Patients and human plasma samples. Maternal plasma samples were obtainedfrom an ongoing investigation of preeclampsia (Prenatal Exposures andPreeclampsia Prevention; PEPP) at the Magee-Women's Research Instituteand Magee-Womens Hospital, University of Pittsburgh, Pa. The PEPP studywas approved by the University of Pittsburgh institutional review boardand informed consent was obtained from all participants. The PEPPcommittee approves the use of these previously frozen samples andde-identified clinical data. Blood samples from all women were collectedinto EDTA plasma separation tubes. Plasma was centrifuged at 1400 to1600 revolutions per minute and aliquoted. Plasma was then pooled from 4groups: nonpregnant women who had never been pregnant (NonP; n=9),pregnant women with uncomplicated pregnancies (NP; n=12), pregnant womenwho developed LPE (n=10) and pregnant women who developed EPE (n=5). Thepooled plasma was stored at −80° C. until experimentation. The womenenrolled in the preeclamptic groups met the criteria according to theAmerican College of Obstetricians and Gynecologists of blood pressuregreater than or equal to 140 mmHg systolic and/or 90 mmHg diastolic plusan increase of greater than 30 mmHg systolic and/or 15 mmHg diastolicplus proteinuria greater than 300 mg/24 hrs or at least equal to 2+protein using a urine dipstick test. EPE women were diagnosed withpreeclampsia and delivered before 34 weeks of gestation and LPE womenwere diagnosed and delivered after 34 weeks of gestation (see Table 1).Only non-smokers and nulliparous women were included during plasmacollection for this study.

Measurement of oxLDL in human plasma samples. The levels of oxLDL in theplasma from the NonP, NP, LPE and EPE women were determined using asandwich Enzyme-Linked-Immuno-Sorbent-Assay (ELISA) Kit(Immunodiagnostik, Bensheim, Germany) according to the manufacturers'instructions. Measurements were performed in triplicate and averaged.

Animals. Female Sprague Dawley virgin nonpregnant rats (12-14 weeks;250-300 grams) or female Sprague Dawley pregnant rats (day 5; 12-14weeks, 250-300 grams) were purchased from Charles River (Saint-Constant,QB, Canada). All of the procedures were approved by the University ofVermont Institutional Animal care and Use Committee and complied withthe National Institutes of Health Guide for the Care and Use ofLaboratory Animals. Animals were housed in the Animal Care facility,which is an Association for Assessment and Accreditation of LaboratoryAnimal Care-accredited facility. Animals had access to food and water adlibitum and were maintained on a 12-hour light/dark cycle.

High-cholesterol rat model. The pregnant rats were divided into 2 groupson day 6 of pregnancy: a late-pregnant control group (LP-CTL; n=8) and alate-pregnant high-cholesterol treated group (LP-HC; n=8). The LP-CTLrats received Prolab® 3000 rodent chow for 14 days. The LP-HC animalsreceived a 14 day diet consisting of Prolab® 3000 rodent chow, including2% cholesterol and 0.5% cholic acid (added to lower hepatic clearance ofcholesterol) to increase total and LDL-cholesterol. Experimentation wasdone on day 14 of the diet that equaled day 20 of pregnancy in allanimals.

Blood pressure measurements. All LP-CTL and LP-HC animals had bloodpressure measurements taken on day 2 and day 13 of their diet. Animalswere trained for 2 days prior to the first day of blood pressuremeasurements to make the rats familiar with handling and restraintassociated with the procedure. This way, we prevented measuringartificially high blood pressures by reducing stress in the animals.Blood pressures were taken using a noninvasive tail cuff method (CODAS8, Kent Scientific, Torrington, Conn.), as previously done (51).Briefly, animals were placed in individual holders on a heating plateand both an occlusion cuff and a volume pressure-recording cuff wereplaced on the tail close to the base. Animals were warmed to 30° C. foroptimal volume pressure recording. Systolic, diastolic and mean bloodpressure, heart pulse rate, tail blood volume, and tail blood flow weremeasured simultaneously.

Rat plasma samples. Plasma samples were obtained from trunk blood fromLP-CTL and LP-HC rats. Plasma was collected in EDTA plasma separationtubes and centrifuged for 10 minutes at 2500 revolutions per minute.Plasma was then aliquoted and directly used for permeabilityexperiments.

BBB permeability measurements. The first set of experiments wasperformed to determine the effects of circulating factors in plasma fromEPE and LPE women on the BBB permeability compared to NP women and NonPwomen. We measured L_(p), the critical transport parameter that relateswater flux to hydrostatic pressure, in isolated cerebral veins fromnonpregnant female rats after perfusing with plasma from the 4 groups ofwomen, as described previously (52). This method of measuring BBBpermeability was specifically developed to have a direct measure ofwater permeability and has been successfully used in several previousstudies (13, 52-54).

The vein of Galen was used for all permeability experiments asrepresentation of the BBB because this vein has BBB properties and iswhere BBB disruption occurs first during acute hypertension (55).Further, only veins from NonP rats were used to isolate the possibleeffects of circulating factors in the plasma and our previous studieshave shown no difference in BBB permeability comparing vessels from NonPand pregnant rats (53). Briefly, cerebral veins were carefully dissectedout of the brain of NonP rats and the proximal end mounted on one glasscannula in an arteriograph chamber. Veins were perfused intraluminallywith 20% v/v plasma from either NonP (n=7), NP (n=7), LPE (n=6) and EPE(n=6) in a HEPES buffer for 3 hours at 10±0.3 mmHg and 37° C. The distalend of the vessel was tied off with a nylon structure. After thisincubation period with plasma, intravascular pressure was increased to25±0.1 mmHg and the drop in pressure due to transvascular filtration ofwater out of the vessel in response to hydrostatic pressure was measuredfor 40 minutes. The decrease of intravascular pressure per minute(mmHg/min) was converted to volume flux across the vessel wall (μm3)using a conversion curve, as previously described (52). After flux wasdetermined, transvascular filtration per surface area (Jv/S) and L_(p)were calculated by normalizing flux to the surface area and oncoticpressure of the plasma perfusate that was determined by a commerciallyavailable oncometer.

In a separate set of experiments, we determined the involvement of LOX-1activation on BBB permeability by adding a neutralizing antibody toLOX-1 (5 μg/ml; n=6) to the plasma from EPE women before perfusing theplasma into the vein of Galen and the permeability experiment wasrepeated.

Another set of experiments was performed to determine the involvement ofONOO⁻ generation in plasma from EPE women by adding the ONOO⁻decomposition catalyst FeTMPyP (50 μM; n=6) to the plasma from EPE womenbefore perfusing the plasma in the cerebral veins. FeTMPyP is a ferricporphyrin complex that catalytically isomerizes peroxynitrite to nitratein vitro (56). It has been shown to be selective for blocking ONOO⁻effects without interfering with NO or superoxide (57). Therefore,FeTMPyP serves as a selective ONOO⁻ decomposition catalyst. In addition,the concentration of 50 μM was based on one of our earlier studies whereFeTMPyP was used in isolated arteries to scavenge ONOO⁻ (58).

A separate set of experiments was performed to determine the effects ofexogenous oxLDL on BBB permeability by the addition of exogenous humanoxLDL (3.5 μg/ml; n=7) to plasma from NonP women before perfusing theplasma in the cerebral veins. Thus, we could determine the direct effectof oxLDL on BBB permeability and compare this to the BBB permeabilitymeasured in the EPE plasma. For these experiments, plasma from NonPwomen was chosen rather than plasma from NP women to eliminate otherpossible circulating factors present in pregnancy that could interactwith oxLDL. To determine if there was a causal link between oxLDLincreasing BBB permeability and ONOO⁻ generation, we repeated theseexperiments with the addition of FeTMPyP (50 μM; n=6) to theoxLDL-plasma mixture before perfusion in the vein of Galen and measuredL_(p).

The last set of experiments was performed to determine the involvementof high levels of cholesterol in pregnancy on pregnancy outcome and BBBdisruption. The cerebral vein of Galen was carefully dissected out of LPrats that received either a control diet or a high-cholesterol diet andmounted in the arteriograph chamber, as described above. For theseexperiments, 20% v/v plasma in HEPES buffer was taken and perfused inveins from the same animals. To determine the contribution of oxLDL inincreasing BBB permeability in the high-cholesterol treated pregnantrats, the same neutralizing LOX-1 antibody (5 μg/ml; n=8) was added tothe plasma from the LP-HC animals before perfusion in the cerebralveins.

Measurement of mRNA expression of LOX-1 using real-time quantitive PCR.The middle cerebral artery (MCA) was used as a representative cerebralvessel to measure mRNA expression of LOX-1 in the LP-CTL and LP-HCanimals, as the Vein of Galen was used of for permeability experimentsin these animals. Total RNA was extracted from MCAs from LP-CTL (n=6)and LP-HC (n=7) rats using Trizol reagent (Life Technologies) followedby purification using an Rneasy Micro Kit (Qiagen) per manufacture'sprotocols. RNA concentrations and quality were determined using anAgilent Bioanalyzer (Agilent). Real time PCR was performed in a two-stepprocess. RNA was reverse transcribed using a mix of oligo dT primers andrandom primers using the iScript cDNA Synthesis Kit (Biorad). For eachsample, cDNA was used to amplify the target gene LOX-1 and two generallyused housekeeping genes, Hprt1 and Ywhaz. Primers were designed by theObstetrics and Gynecology Departmental Molecular Core Facility at theUniversity of Vermont using PrimerSelect (DNASTAR). The quantitative PCRprimers for the rat transcripts were: LOX-1 (f):-GATGATCTGAACTTCGTCTTACAAGC-(SEQ ID NO. 1) and (r):-TCAGCAAACACAACTCCTCCTT-(SEQ ID NO. 2); and the housekeeping genes Hprt1(f): -CTCATGGACTGTTATGGACAGGAC-(SEQ ID NO. 3) and (r):-GCAGGTCAGCAAAGAACTTATAGCC-; (SEQ ID NO. 4) Ywhaz (f):-GATGAAGCCATTGCTGAACTTG-(SEQ ID NO. 5) and (r):-GTCTCCTTGGGTATCCGATGTC-(SEQ ID NO. 6). One microliter of cDNA was usedper reaction with 150 nM of the forward and reverse primers and 12.5 μlof Power Sybrgreen Master mix (Life Technologies) in a 25 μl reaction.The reactions were performed using an initial denaturation of 3 minutesat 95° C., 40 cycles of 15 seconds at 95° C. and 60 seconds at 60° C.,followed by a melt curve analysis to ensure only the correct product wasamplified. One set of PCR products for each gene were checked forcorrect size on a 2% Agarose gel. Each sample was run in triplicate onthe ABI 7000 Sequence Detection System (ABI). For each primer set in thereal time PCR reaction, negative water controls were performed to ensureno contamination in the reagents as well as no secondary primerstructures were amplified. The LOX-1 primer was designed to span anexon-exon junction to make sure that genomic DNA was not amplified.

Drugs and solutions. HEPES physiological salt solution was made freshdaily and consisted of (mmol/L): 142.00 NaCl, 4.70 KCl, 1.71 MgSO4, 0.50EDTA, 2.80 CaCl2, 10.00.

HEPES, 1.20 KH2PO4, and 5.00 dextrose. FeTMPyP was purchased fromCalbiochem, (Gibbstown, N.J., USA; 341501). LOX-1 antibody was purchasedfrom R&D systems R&D systems (Minneapolis, Minn., USA; AF1564). OxLDLwas purchased from Kalen Biomedical (LLC, Montgomerey Village, Mn, USA;770252-7).

Statistical analysis. Data are presented as mean±standard error of themean. Analyses were performed by one-way ANOVA with a post-hoc StudentNewman Keuls test for multiple comparisons where appropriate or with aStudent's T-test. Differences were considered statistical significant atP<0.05.

RESULTS Example 1: Examining Patient Characteristics

There were no significant differences between the 4 groups of women withrespect to age or BMI. Blood pressure measured before 20 weeks ofgestation was similar in all pregnant groups and was comparable to theblood pressure measured in the group of women who had never beenpregnant (NonP). At time of delivery, blood pressure measured in womenwith an uncomplicated pregnancy (normal pregnant; NP) had not changedcompared to the blood pressure measured before 20 weeks of gestation.However, blood pressure measured at delivery from both preeclampticgroups was significantly higher compared to their blood pressuremeasured before 20 weeks of gestation. Blood pressure was notsignificantly different between EPE and LPE women. The gestational ageof delivery was significantly higher for the NP women compared to bothof the preeclamptic groups. The gestational age of delivery was alsosignificant lower in EPE women compared to the LPE women. Lastly, thebirth weight of the infants born was significantly different between the3 pregnant groups with the EPE women having babies with the lowest birthweight and the lowest birth weight percentile compared to the othergroups of pregnant women (summarized in Table 1).

TABLE 1 NonP NP LPE EPE (n = 9) (n = 12) (n = 10) (n = 5) Age (yrs) 25.4(±4.6) 26 (±4.5) 31 (±3.6) 25 (±5) BMI 24.0 (±3.2) 26.4 (±3.7) 26.5(±7.4) 24.3 (±3.0) GA sample (wks) — 34.9 (±1.8) 35.8 (±1.6) 32.3(±1.6)* GA delivery (wks) — 40.1 (±0.93)^(#) 36.0 (±1.4){circumflex over( )} 32.5 (±1.4)* BP (sys) <20 wks  107 (±6.8) 110.1 (±7.2) 119.5(±12.4) 122.0 (±5.6) BP (dias) <20 wks 65.5 (±5.5) 66.8 (±3.9) 73.0(±7.3) 73 (±4) BP (sys) delivery — 119.4 (±7.3)^(#) 152.40 (±8.59) 160.0(±9.5) BP (dias) delivery — 68.8 (±11.7)^(#) 91.70 (±6.43) 100.6 (±11.0)Birthweight (g) — 3601 (±380)^(#) 2271.3 (±143.2){circumflex over ( )}1462 (±223)* *P < 0.05 vs NP and LPE. ^(#)P < 0.05 vs LPE and EPE.{circumflex over ( )}P < 0.05 vs NP and EPE. Abbreviations: NonP =nonpregnant; NP = normal pregnant; LPE = late-onset preeclampsia; EPE =early-onset preeclampsia

TABLE 2 LP-CTL LP-HC (n = 8) (n = 8) Weight (grams) 408 (±6) 403 (±10)BP (day 13 diet; mmHg) 97 (±2) 104 (±2)* Pups (#) 16 (0.5) 12 (±0.6)*Resorptions (#) 0.7 (±0.2) 1.4 (±0.4) Avg weight pups (grams) 2.42(±0.06) 2.12 (±0.04)* Avg weight placenta (grams) 0.44 (±0.01) 0.48(±0.01)* *P < 0.05 vs. LP-CTL Abbreviations: LP-CTL = late-pregnantcontrol rats; LP-HC = late-pregnant high cholesterol treated rats

Example 2: The Effect of Circulating Factors in Plasma from EPE Women onBBB Permeability

It was determined whether circulating factors in plasma from EPE womenhave different effects on BBB permeability in cerebral veins compared toplasma from LPE women. Here 20% v/v plasma was perfused from LPE and EPEwomen in a cerebral vein from NonP rats and several parameters weremeasured including flux and Jv/s to determine L_(p). Plasma from NonPand NP women were used as controls. The decrease in intravascularpressure due to filtration in response to plasma from EPE, LPE, NP andNonP women was significantly greater in all pregnant groups compared toNonP women (FIG. 2A). Importantly, the decrease in intravascularpressure was significantly greater in EPE women compared to all othergroups. After converting the intravascular pressure drop into actualvolume flux across the vessel wall (FIG. 2B), plasma from both LPE andEPE showed a significantly increase in flux compared to plasma from NonPwomen. However, plasma from EPE women caused a significantly higher fluxcompared NonP, NP and LPE women. After normalizing volume flux tosurface area and oncotic pressure of the plasma perfusate (FIGS. 2C-D),plasma from both LPE and EPE women still significantly increased BBBpermeability compared to plasma from NonP women. Also, circulatingfactors in plasma from EPE women significantly increased Jv/S and L_(p)and thereby increased BBB permeability compared to all other groups,including LPE women. Thus, plasma from EPE women caused a greaterincrease in BBB permeability compared to plasma from LPE women that mayunderlie neurological complications.

Example 3: The Involvement of oxLDL and LOX-1 Activation in IncreasedBBB Permeability in EPE Plasma

To determine if oxLDL levels are increased in EPE women that could beinvolved in BBB disruption, oxLDL levels were measured from the plasmaof all 4 groups of women. Plasma from EPE women had a 260% increase inoxLDL levels compared to plasma from LPE women (FIG. 3A). The level ofoxLDL in plasma from NonP, NP and LPE women were comparable. Thus, asignificant increase in the amount to oxLDL in plasma from EPE vs. LPEwomen.

OxLDL binds its receptor LOX-1 that, once activated, causes endothelialdysfunction in many pathological states such as atherosclerosis,diabetes and hypertension (19, 20). To determine if LOX-1 activation isinvolved in increasing BBB permeability in cerebral veins in response toplasma from EPE women, LOX-1 was neutralized and L_(p) then determined.Since Lp is a critical parameter of BBB permeability, only L_(p) at 36minutes is in the subsequent figures as a bar chart. Adding aneutralizing LOX-1 antibody in the plasma from EPE women beforeperfusion in the vein of Galen abolished the increased BBB permeabilityinduced by circulating factors in this plasma (FIG. 3B). Thus, LOX-1activation is involved in increasing BBB permeability in plasma from EPEwomen.

Example 4: Effect of oxLDL on BBB Permeability

Next, to determine if oxLDL is able to increase BBB permeability withoutthe presence of other circulating factors present in the plasma from EPEwomen, 3.5 μg/ml of purified oxLDL was added to plasma from NonP womenbefore perfusing the plasma in cerebral veins. This concentration wasused based on the values in the EPE plasma measured by ELISA.Importantly, addition of exogenous oxLDL to plasma from NonP womencaused a significant increase in BBB permeability that was comparable toplasma from women with EPE (FIG. 3C). Thus, these data suggest that highlevels of oxLDL in plasma from EPE women increase BBB permeabilitythrough LOX-1 activation.

Example 5: Effect of FeTMPyP on oxLDL-LOX-1 Induced BBB Permeability inEPE Plasma

Activation of LOX-1 leads to increased production of superoxide throughNADPH-oxidase that rapidly binds NO to form ONOO⁻ (25). Previous studiesconfirmed generation of ONOO⁻ in the placental and maternal systemicvasculature of preeclamptic women, however, involvement of ONOO⁻generation in response to LOX-1 activation in disrupting the BBB is notknown (29, 31). Cerebral veins from NonP rats were perfused with plasmafrom EPE women plus 50 μM FeTMPyP, a selective ONOO⁻ decompositioncatalyst. FeTMPyP significantly inhibited BBB permeability caused byplasma from EPE women (FIG. 4A), demonstrating that ONOO⁻ generation isalso involved in BBB disruption in cerebral veins after exposure toplasma from EPE women.

Example 6: Effect of Exogenous oxLDL on BBB

To determine that the increase in BBB permeability caused by exogenousoxLDL (FIG. 3C) was due to increased generation of ONOO⁻, 50 μM FeTMPyPwas added to the plasma from NonP women plus oxLDL. Addition of FeTMPyPinhibited BBB permeability induced by oxLDL (FIG. 4B), demonstratingthat ONOO⁻ generation is induced by oxLDL and leads to BBB disruption.

Example 7: Effects of Pathologically High Lipids on Blood Pressure,Pregnancy Outcome, BBB Permeability and LOX-1 mRNA Expression in aPregnant High-Cholesterol Rat Model

Because increased levels of cholesterol are present in preeclampsia(14-16) and increased oxLDL is involved in disrupting the BBB in EPE, itwas important to determine the effect of pathologically high levels ofcholesterol in pregnancy on pregnancy outcome and BBB permeability usinga rat model. Rats treated with high-cholesterol for 14 days had a 350%increase in cholesterol compared to LP-CTL animals. Table 1 showscharacteristics and pregnancy outcome in these rats. LP-HC rats hadmodest, but significantly higher blood pressures vs. LP-CTL rats.Further, LP-HC rats carried a significantly smaller number of pups, andhad a higher rate of reabsorptions. Pup weights from the LP-HC rats weresignificantly lower, similar to EPE, with a significant increase inplacental weight.

Example 8: Effect of Pathologically High Cholesterol in Pregnant Rats onBBB Permeability

To determine if pathologically high levels of cholesterol in pregnantrats increased BBB permeability, cerebral veins from LP-HC or LP-CTLrats were perfused with 20% v/v plasma from the same animal and measuredL_(p). BBB permeability in cerebral veins from LP-HC animals wassignificantly increased compared to the LP-CTL animals (FIG. 5A). Todetermine if LOX-1 activation was involved in increasing BBBpermeability in LP-HC animals, a neutralizing LOX-1 antibody was addedto the plasma from LP-HC rats before perfusing in the cerebral veins.Addition of the LOX-1 antibody abolished the increased BBB permeabilityin LP-HC animals (FIG. 5A); suggesting oxLDL was involved in increasingBBB permeability in the LP-HC animals, similar as in EPE.

Example 9: LOX-1 mRNA Expression Levels in Cerebral Vasculature as anIndependent Factor of BBB Permeability

Lastly, to determine if LOX-1 mRNA expression was increased in thecerebral vasculature of LP-HC animals that may increase BBB permeabilityindependent of increased oxLDL, we isolated the MCAs from the sameanimals as used for the permeability experiments to determine mRNAexpression of LOX-1. mRNA expression of LOX-1 was similar in the LP-HCand LP-CTL animals (FIG. 5B), suggesting that circulating oxLDL isactivating LOX-1 to increase BBB permeability as opposed to upregulationof LOX-1.

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Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

What is claimed is:
 1. A method for treating a subject comprising:administering to a pregnant subject at risk of seizure having elevatedlevels of oxLDL, a LOX-1 inhibitor, wherein the LOX-1 inhibitor is anantibody or a small molecule, in an effective amount to treat thesubject.
 2. The method of claim 1, wherein the subject does not have aninflammatory disorder.
 3. The method of claim 1, wherein the subject hasnot been diagnosed with an inflammatory disorder.
 4. The method of claim1, wherein the LOX-1 inhibitor comprises a statin or a procyanidin.